Acute lymphoblastic leukemia (ALL) is cancer of white blood cells. Approximately 4,000 new cases of ALL are diagnosed annually in the US alone, with 60% of them found in children. One of the major drugs used in ALL treatment is L-asparaginase (ASNase), which induces a systemic depletion of asparagine (ASN); an essential nutrients for ALL cells. Nevertheless, clinical use of ASNase encounters two major setbacks. First, ASNase is a non-human, immunogenic protein, and its clinical use is thus associated with major anaphylactic responses. Secondly, like most protein drugs, ASNase is susceptible to proteolytic degradation and RES clearance. As a result, plasma half-life of ASNase is rather short (~25hr), thereby demanding frequent injections of the drug that further increase the risk of allergic attack. To overcome such problems, efforts have been focused on protection of ASNase with a synthetic or natural carrier. Among these carrier systems, red blood cells (RBC) appear to be most appealing, because they are biocompatible, biodegradable, and also possess an unmatched life-span of ~120 days. A variety of techniques has been attempted to encapsulate proteins into RBC. However, all of these methods require disruption of RBC membrane with a chemical or physical force to create pores for proteins drugs to diffuse in. Unfortunately, insult on the RBC surface by such an invasive force causes membrane deterioration and, consequently, results in a loss of structural integrity and cellular components of the RBC, rendering it prone to destruction by the host immune system. It should be noted that in order to inherit the benefits of RBC as a long-lasting, natural carrier, it is essential to retain both structural and functional integrity of RBC. Yet, all of the existing RBC encapsulation techniques fail to recognize this critical aspect. Recently, a family of potent cell-penetrating peptides (CPP) has been discovered. In vitro and in vivo results revealed that, by covalently linking CPP to almost any type of cargos including proteins, PTD was able to ferry the attached species across cell membranes of all tissue types, including the brain. Remarkably, PTD-mediated cell entry does not induce any membrane perturbation or alteration. These desirable properties provide the conceptual framework of the proposed non-invasive, RBC-encapsulation technology for ASNase. Briefly, ASNase will be covalently linked with a PTD peptide (i.e. LMWP) via a disulfide linkage. Due to the potent cell-penetrating activity of LMWP, the LMWP-ASNase conjugates should be able to internalize a RBC without altering its structural and functional attributes. Within the cell, LMWP would be dissociated from ASNase via degradation of the disulfide bond, due to the presence of a high level of cytosolic reductase activity. This bond dissociation would allow ASNase to remain permanently entrapped within RBC, ensuring a full protection of ASNase from detection and destruction by the host immune system. Hence, the ASNase-encapsulated RBC would function as a live bioreactor, depleting ASN from the circulation and depriving ALL cells of essential nutrients, subsequently leading to their deaths. If both of the physical and biological attributes of RBC can be fully retained after encapsulation, the entrapped ASNase would then accede to the same life-span of native RBC (120 days), yielding the longest lasting therapeutic effects than any current ASNase therapies. This would reduce current ASNase dosing frequency by more than 100 folds, significantly alleviating the toxic side effects associated with present ASNase therapies. Extremely promising preliminary results have been obtained, which showed RBC processed by this novel technology exhibited an intact structure and functionality that were indistinguishable from normal RBC. In vivo results also showed that RBC-entrapped ASNase not only inherited a prolonged plasma half-life in healthy mice but also displayed a long-lasting therapeutic effects in ALL-harboring mice. In this Phase I research, we plan to build on these exciting preliminary findings and carry out a proof-of-concept animal investigation to further validate this technology. Our ultimate goal is to develop this RBC- ASNase technology into a real clinical remedy. PUBLIC HEALTH RELAVENCE:One of the major drugs used in leukemia treatment requires demanding and frequent injections of the drug during clinical application that increases the risk of allergic attack. There is a great need to enhance current leukemia therapy while minimizing harm to the patient. In this project we will utilize novel peptides that can internalize the drug in red blood cells as a delivery agent and reduce the dose required to treat leukemia by 100 fold. [unreadable] [unreadable] [unreadable]